• <tr id="yyy80"></tr>
  • <sup id="yyy80"></sup>
  • <tfoot id="yyy80"><noscript id="yyy80"></noscript></tfoot>
  • 99热精品在线国产_美女午夜性视频免费_国产精品国产高清国产av_av欧美777_自拍偷自拍亚洲精品老妇_亚洲熟女精品中文字幕_www日本黄色视频网_国产精品野战在线观看 ?

    Determination of inhibitory activity of Salvia miltiorrhiza extracts on xanthine oxidase with a paper-based analytical device

    2021-11-11 13:37:50XingchuGongJingyuanShaoShangxinGuoJingjingPanXiaohuiFan
    Journal of Pharmaceutical Analysis 2021年5期

    Xingchu Gong , Jingyuan Shao , Shangxin Guo, Jingjing Pan, Xiaohui Fan

    Pharmaceutical Informatics Institute, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China

    Keywords:Paper-based analytical device (PAD)Point-of-care testing Xanthine oxidase Salvia miltiorrhiza extract 3D printing

    ABSTRACT A novel paper-based analytical device (PAD) was prepared and applied to determine the xanthine oxidase(XOD)inhibitory activity of Salvia miltiorrhiza extracts(SME).First,polycaprolactone was 3D printed on filter paper and heated to form hydrophobic barriers. Then the modified paper was cut according to the specific design. Necessary reagents including XOD for the colorimetric assay were immobilized on two separate pieces of paper. By simply adding phosphate buffer, the reaction was performed on the double-layer PAD. Quantitative results were obtained by analyzing the color intensity with the specialized device system (consisting of a smartphone, a detection box and sandwich plates). The 3Dprinted detection box was small,with a size of 9.0 cm×7.0 cm×11.5 cm.Color component G performed well in terms of linearity and detection limits and thus was identified as the index. The reaction conditions were optimized using a definitive screening design.Moreover, a 10% glycerol solution was found to be a suitable stabilizer.When the stabilizer was added,the activity of XOD could be maintained for at least 15 days under 4 °C or-20 °C storage conditions.The inhibitory activity of SME was investigated and compared to that of allopurinol. The results obtained with the PAD showed agreement with those obtained with the microplate method.In conclusion,the proposed PAD method is simple,accurate and has a potential for point-of-care testing. It also holds promise for use in rapid quality testing of medicinal herbs, intermediate products, and preparations of traditional Chinese medicines.

    1. Introduction

    From ancient times until today,Chinese people have used herbal drugs for health maintenance and prevention and treatment of various diseases.The effectiveness and safety of traditional Chinese medicines(TCMs)are key factors in the quality control of TCMs[1].At present, chemical composition analysis is used in the quality control of many TCMs [2,3]. However, analytical methods for the separation of the complex components of TCMs are sometimes difficult to develop,and typically require specific instruments,such as high-performance liquid chromatography (HPLC) and gas chromatography (GC) [4,5], which also makes detection costly. Moreover, TCMs present pharmacological effects through multiple targeting and integrative adjusting [6-8]. As a result, it is usually difficult to determine the internal effectiveness of TCMs just by identifying several of their chemical constituents. Therefore, the bioactivity of TCMs may be used as quality indices[9,10].Biological activities can be evaluated in cell,tissue,and animal models,which suffer from the disadvantages of large capital costs and long operation time. In conclusion, the development of effective and rapid methods for estimating the bioactivity of TCMs is of practical significance.

    Currently, paper-based analytical devices (PADs) are gaining increasing attention worldwide due to their specific advantages of good portability, easy operation, reasonable cost, and rapid detection [11]. The method utilizes hydrophilic paper as a substrate material and creates hydrophobic barriers on it in order to generate functional areas. Common fabrication methods for PADs mainly include wax patterning[12],inkjet printing[13],photolithography[14],and paper cutting[15].Most of these methods are simple,fast and capable of mass production. When reagents and samples are spotted on the test area, they can react and produce color or fluorescence, which can be quantitatively detected. Among the different types of signals, colorimetric signals are most commonly used owing to their simplicity,and the analysis is usually combined with practical and inexpensive equipment such as smartphones and scanners [16]. In biomedical applications of PADs, various approaches are adopted to generate color,including enzymatic assays[17], immunoassays [18], and nanoparticle-based assays [19]. To date, PADs have been widely applied in the concentration determination of many analytes,such as glucose[20],uric acid[21],and haptoglobin [22]. In general, PADs show potential applications in point-of-care (POC) testing according to the requirements of the ASSURED criteria, which dictate that POC tests should be affordable,sensitive,specific,user-friendly,rapid and robust,equipmentfree,and deliverable to end-users [23,24].

    There are several works on the activity determination of foods.Sharpe et al. [25] established a paper-based colorimetric method based on metal oxide nanoparticles to analyze antioxidantcontaining samples in terms of their gallic acid equivalents.Nuchtavorn et al. [26] presented a novel method to create paperbased microfluidic devices (μPADs) using a desktop digital craft plotter/cutter and technical drawing pens. They used PADs to measure the flavonoid and phenolic content and DPPH free radical scavenging activity in beverages including tea, wine, and beer samples.

    There are also some reports on TCM activity determination with PADs. In a previous work, PADs were used to analyze the antioxidant activity of Danhong injection and its intermediates,providing a method to determine the biological activities of TCMs and their intermediates [27]. In another previous work, the inhibitory activity of mulberry extracts on α-glucosidase was also determined using PADs [28]. When PADs are integrated with bioassays for TCMs, there are several advantages. First, PADs can be used as a platform to measure the effects of TCMs on target enzymes, thus reflecting the efficacy of TCMs from a holistic perspective.Second,PADs have their own characteristics, such as low cost, easy operation, and convenient result readout, indicating that they are appropriate for performing bioassays for TCM quality evaluation.However, portable analytical systems for TCM activity determination have not been reported.

    In this work, a portable PAD for the analysis of enzyme inhibitory activity of xanthine oxidase (XOD) was developed. XOD catalyzes the oxidation of hypoxanthine and xanthine to uric acid.Therefore,it is considered a therapeutic target for gout, which is a form of inflammatory arthritis characterized by tissue damage and associated with a high level of uric acid [29]. It was reported that many herbs, includingSalvia miltiorrhiza radix,Rhei radix,Polygoni cuspidati rhizoma,Selaginellae herba,Paeoniae radix rubra, andGinkgo folium, have anti-inflammatory effects and inhibit XOD activity [30]. Therefore,Salvia miltiorrhizaextracts (SME) were studied for their XOD inhibitory activity.

    In this work,polycaprolactone-modified paper was fabricated via 3D printing and heating.Then,the modified paper was manually cut and functionalized with XOD, which could react with xanthine and nitrotetrazolium blue chloride(NBT)to induce a color change.A PAD with a double-layer structure was fabricated.A suitable color index was selected, and the reaction conditions were optimized by a definitive screening design. The best stabilizer was selected.Subsequently,the PAD withthe optimum conditions wasapplied to analyze the bioactivities of SME samples,and the results were compared with those obtained via the conventional microplate method.The advantages of the present PAD method were also discussed.

    2. Materials and methods

    2.1. Materials

    NBT (98%) and allopurinol (98%) were purchased from Aladdin Industrial Corp.(Shanghai,China).XOD and xanthine(≥99%)were purchased from Sigma-Aldrich Chemical Corp.(St.Louis,MO,USA).Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA-2Na·2H2O, ≥99.0%), dipotassium hydrogen phosphate trihydrate(K2HPO4·3H2O, ≥ 99.0%), potassium dihydrogen phosphate(KH2PO4,≥99.5%),and dextran(Mw 20,000)were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Glycerol(99%) was purchased from Shanghai Macklin Biochemical Co., Ltd.(Shanghai, China). Bovine serum albumin (BSA) was purchased from Absin Bioscience Inc.(Shanghai,China).Trehalose(≥99%)was purchased from Sinozyme Biotechnology Co.,Ltd.(Nanning,China).Ultrahigh-purity water was produced using a Milli-Q water purification system from Millipore(Milford,MA,USA).The 3D printing materials, namely, polycaprolactone, wax wire, and polylactic acid(PLA) filament, were purchased from Dongguan Top Cool Electronics Technology Co., Ltd. (Dongguan, China), Fuzhou Zhilei Electronic Technology Co., Ltd. (Fuzhou, China), and Hangzhou Shining 3D Technology Co., Ltd. (Hangzhou, China), respectively.Salvia miltiorrhiza(batch No.170901) was purchased from Jiuzhou Drugstore(China Jo-Jo Drugstores Inc., Hangzhou, China).

    2.2. Reagent preparation

    The XOD solution was dissolved in phosphate buffer (PB), and the enzyme concentration was optimized.The buffer pH value was calibrated with a pH meter (SevenMulti, Mettler Toledo, Schwerzenbach, Switzerland) and adjusted to various values (7.0, 7.5 and 8.0) before use. Substrate solutions of xanthine (0.48 mM) and a stock solution of allopurinol(400 μg/mL)were prepared separately in the same PB solution. Allopurinol solutions with serial concentrations (10-100 μg/mL) were obtained by diluting the stock solution with an appropriate amount of the buffer solution.A 10%(V/V) glycerol solution was prepared with ultrahigh-purity water.

    2.3. Sample preparation

    First, 50 g ofSalvia miltiorrhizawas extracted twice with water(first with 350 mL for 30 min and then with 300 mL for 20 min).Then, the extracts were merged for rotatory evaporation and concentrated to 250 mL. To prepare the samples for testing, PB solution (pH 8.0) was used to quantitatively dilute the SME samples.

    2.4. Preparation of PAD

    In brief, filter paper was first modified with polycaprolactone and then folded into a double-layer construction with simple paper cutting and origami steps. A schematic illustration of the fabrication process is presented in Fig. S1. A predesigned pattern of the paper-based chip was drawn with 3D Builder software (Microsoft Corporation, USA), and the top view of the designed pattern is shown in Fig. S1A. Then, polycaprolactone was printed on Whatman No.202 quantitative filter paper(Hangzhou Wohua Whatman Filter Paper Co., Ltd., Hangzhou,China) using a desktop 3D printer(Einstart-S, Hangzhou Shining 3D Technology Co., Ltd., Hangzhou,China), as shown in Fig. S1B. Next, the printed paper-based chip was placed into an electric baking pan(JK-3030S2,Joyoung Co.,Ltd.,Hangzhou, China) and heated at 150°C for 90 min. Upon heating,polycaprolactone melted and penetrated through the paper to form hydrophobic barriers, as shown in Fig. S1C. After that,polycaprolactone-modified paper was cut to obtain two pieces, as shown in Fig. S1D.

    The reagents were loaded onto the hydrophilic regions of the paper-based chips, as shown in Fig. S1E. A total of 20 μL of XOD solution and a certain volume of 10% glycerin solution were pipetted onto the reaction zone of one piece of paper (enzyme layer). After drying at room temperature for 30 min, XOD was immobilized on the reaction region. A certain volume of xanthine solution and 20 μL of NBT were preloaded onto the reaction zone of another piece of paper(substrate layer)and air dried.After that,the PAD was obtained and available for the sample test.

    2.5. Colorimetric reaction

    The practical operation of the colorimetric reaction based on the PAD is displayed in Fig.1.The two pieces of the PAD were combined to form a double-layer reaction structure in which the substrate layer was the upper layer,as shown in Fig.1A.Afterwards,the PAD was mounted on a small support plate with a lid and several clips to clamp it.All of the reaction devices were made by 3D printing with wax wire as the printing material,as shown in Figs.1B and C.When the reaction process was performed, since the enzyme and substrate solutions had already been immobilized onto the PAD during fabrication, the only reagent that needed to be added was buffer solution. Therefore, a certain volume of PB solution was added to the substrate layer so that it could penetrate down and accelerate the process of the enzymatic reaction. Once the reaction was completed, the substrate layer was separated from the enzyme layer, and then the paper-based chip was dried. The enzyme layer with a deeper color was selected to analyze the color intensity,while the substrate layer with a lighter color was unsuitable for color analysis, as shown in Fig.1A.

    2.6. Determination of colorimetric reaction results

    After the enzymatic reaction was carried out,the color intensity of the enzyme layer was measured with two detection systems.The first is a homemade detection system we published previously[28].The detailed information of this system can be found in Fig. S2.

    Since the main purpose of this work was to provide a potential method for POC use, a detection device with a smaller size(9.0 cm×7.0 cm×11.5 cm)was designed and 3D printed with PLA filament,as presented in Figs.2A-D.With the goal of reducing the size and weight of the detection box, no LED light tubes were installed in it.Instead,flashlight of the smartphone was turned on to illuminate the paper-based chip.Moreover,the paper-based chip was fixed on the sandwich plates,and they were moved integrally so that the color data of each detection zone could be measured one by one.

    The color intensity was quantified by an app(Color Grab,version 3.6.1,Loomatix Ltd.).It was designed to capture and digitize colors from the real-world to the color intensity of many color models(RGB,CMYK,HSV,etc.)instantly,which made it portable to analyze the results of the color reaction.The software run shot is shown in Fig. 2D. The average intensity of the circular area was obtained.Then,samples were analyzed using both detection systems,and the results were compared.

    Experiments were carried out under a specific combination of conditions to determine the color index for purple.A total of 20 μL of XOD solution(0.05 U/mL)and 10 μL of 10%glycerin solution were spotted onto the enzyme layer and air dried at room temperature.Then,10 μL of allopurinol in a serial concentration gradient(10,20,40,60,and 80 μg/mL)was added separately and dried.Accordingly,10 μL of PB solution(pH 7.5)was added as a blank control.For the substrate layer, 60 μL of xanthine (0.48 mM) and 20 μL of NBT(1.0 mg/mL)were dropped onto it and dried as well.Once the two layers were overlapped as shown above, 20 μL of PB solution (pH 7.5) was added to the substrate layer to trigger the enzymatic reaction.The paper-based device was placed at room temperature for 30 min to promote the reaction. Afterwards, the two layers were separated and allowed to dry at room temperature.

    2.7. Stability experiments

    Fig.1.Overall view of the reaction devices. (A) Practical demonstrations of colorimetric reactions based on PADs. The substrate layer and enzyme layer were overlapped after the reagents were added to each layer and dried.Then,PB solution was added to each test zone to trigger the enzymatic reaction.After the reaction,the two layers were separated,and the enzyme layer had a deeper color than the substrate layer. (B) All the device components were produced by 3D printing. (C) Demonstration of device operation while the enzymatic reaction was taking place.

    Fig.2.Homemade detection devices and color capture process conducted with Color Grab software.(A)Device designed for POC use,(B)the sandwich plates,(C)the paper-based chip was placed on the sandwich plates and inserted into box,(D)the software was used to read the color intensity data in the detection zones,and(E)screenshot of the software while reading color intensity data.

    BSA, dextran, trehalose, and glycerol have been reported as enzyme stabilizers for PADs[31,32].Regarding the stability of XOD immobilized onto the reaction zones,experiments were carried out to screen a suitable stabilizer among BSA(5 mg/mL),dextran(2 mg/mL), trehalose (2.75 mg/mL) and 10% glycerol. First,10 μL of solutions of different stabilizers were added to the circular hydrophilic paper-based zone. Then, 10 μL of XOD (0.05 U/mL) was added to each well. The mixed solution was air dried at room temperature for approximately 30 min.Afterwards,the paper-based chips with immobilized enzyme were separately stored in Petri dishes in two environments,one at room temperature(approximately 27°C)and the other at 4°C. Another set of paper-based chips containing stabilizers and XOD were kept frozen at-20°C.Paper-based chips in which the stabilizers were replaced with PB solution were prepared as controls. These chips were stored under these three conditions at the same time and tested for XOD activity.The activity of the immobilized XOD was tested after being stored for 1,3,5 and 7 days.First,20 μL of xanthine(0.48 mM)and 10 μL of NBT(1.0 mg/mL)were added to each zone and left to react at room temperature for 30 min.Color component G was used to evaluate the stability of XOD. After the initial screening experiments, the activity of XOD with the best stabilizer was tested within 21 days.

    2.8. Experimental design for the optimization of reaction conditions

    Many factors may influence the results of the colorimetric reaction. The main influencing factors were investigated, including XOD concentration(X1),10%glycerol volume(X2),xanthine volume(X3), NBT concentration (X4), PB solution volume (X5), PB solution pH (X6), reaction time (X7), reaction temperature (X8), and drying temperature(X9).The coded and uncoded values of each parameter are listed in Table 1. A 25-run definitive screening design was employed to optimize the reaction parameters, as shown in Table S1. According to preliminary experiments, the concentrationor volume of the main reagents was fixed as follows:XOD volume of 20 μL, xanthine concentration of 0.48 mM, and NBT volume of 20 μL.

    Table 1 Parameters and their levels for the definitive screening design.

    2.9. Data analysis

    Analysis of variance(ANOVA)was used to analyze the results of the definitive screening design. Quadratic models were built to evaluate the effects of parameters on each response according to Equation (1). The corrected Akaike information criterion (AICc)with backward selection was used to help simplify the model. The analysis of the results was performed using Design Expert software(version 10.0.4, Stat-Ease Inc., USA).

    Fig.3.Effects of allopurinol concentration on color intensity in the detection zones.(A)Photograph of the enzyme layer,and(B)scatter graph of color index values with allopurinol concentrations ranging from 0 to 80 μg/mL.

    whereYrepresents the measured values of the response, which is color component G in the RGB color model;a0is a constant;ai,aiiandaijrepresent the regression coefficients for the linear,quadratic,and interaction terms, respectively; andXrepresents each parameter. By applying multivariate regression analysis, an optimized condition was obtained and adopted for subsequent determination of the inhibitory effects of allopurinol and samples on XOD.

    2.10. Model validation and method application

    Three operation points were selected for model validation experiments to ensure that the measured response values agreed with the predictive results obtained from Equation (1). The experimental conditions are presented in Table S2. Subsequently, the optimum method was applied to quantitatively measure the XOD inhibitory activity of SME. Allopurinol solution was used as a positive control. After the addition of 10% glycerol and XOD,10 μL of sample or allopurinol solution was separately dropped onto the enzyme layer and then air dried. The color intensity was closely related to the content of inhibitors,and the purple color decreased as the inhibitor content increased. Therefore, a calibration curve was established by plotting the color intensity values versus the inhibitor concentrations. The limit of detection (LOD) and limit of quantitation(LOQ)were separately calculated by Equation (2)and Equation (3) using OriginPro 2018 (OriginLab Corp., Northampton,MA, USA).

    where σ is the standard deviation of the response and S is the slope of the calibration curve. After that, the inhibitory effects of the samples were compared to that of allopurinol at certain concentrations.

    2.11. Active component of SME and selectivity of the PAD method

    To verify the active component of SME, the XOD inhibitory capacity of salvianolic acid B solution was tested by the optimized PAD method.And the concentration of salvianolic acid B solution was the same as that of SME.On the other hand,SME contains high levels of sugars,mainly including glucose,fructose,and sucrose[33].And it is not reported that sugars can inhibit XOD. Therefore, in order to evaluate the selectivity of the PAD method, the XOD inhibitory capacity of the mixed solution consisting of 0.624 mg/mL fructose,1.152 mg/mL glucose, and 1.564 mg/mL sucrose was studied. Sugar contents of SME were consulted from the literature[33]. Moreover,PAD without any sample solutions served as blank control.

    3. Results and discussion

    3.1. Preparation of polycaprolactone-modified paper

    In this work,to prevent the colorimetric reaction of the enzyme and substrate during the storage of the PAD, the enzyme and substrate were added to two different hydrophilic zones and dried in advance. After the samples interacted with the enzymes, the two reaction zones were overlapped, and the reaction began when the buffer was added.In another design of the paper-based chip,in the center of the rectangle, the hydrophobic barrier was a hydrophilic enzyme reaction area,as shown in Fig.S3A.After these two pieces were folded and brought into contact with each other to form a square reaction area, as shown in Fig. S3B, the liquid tended to overflow when the PB solution was added.

    To avoid sample leakage, an improved design pattern was developed before analysis of SME samples,as illustrated in Fig. S1.Detailed views of the present paper-based chip design are shown in Fig.S4.The basic building block of the hydrophobic barriers was an asymmetrical T-shaped structure, as shown in Fig. S4A. The blank space on one side was a defined square with a side length of 6.0 mm, while the other side was to be cut off, as displayed in Fig.S4B.Every two pieces of these blocks were combined together,and the polycaprolactone-covered boundary was made as an incomplete rectangle. Therefore, the center region without polycaprolactone was the reaction zone. Drawings of the combination of the two sheets of the PAD are shown in Fig.S4C.With this design,additional sample liquid could be added without risking exudation,thus improving the detection sensitivity of the PAD.

    Fig.4.Influence of stabilizers on the stability of XOD in two storage environments over 7 days. (A) Stored at 4 °C, and (B) stored at -20 °C.

    3.2. Selection of color index

    The effects of allopurinol solutions with different concentrations on the color space parameters (RGB, CMYK and grayscale) were investigated. A photograph of the paper-based chip after the colorimetric reaction and the color intensity variations are shown in Fig.3.For samples with higher XOD inhibitory activity,a weaker color was observed in the detection areas. With increased allopurinol concentration, the R value increased from 107 to 180, while the G value increased from 94 to 166.These two responses showed a consistent change trend. Other indexes (including B, C, M and grayscale value)varied within smaller ranges.The Y value remained near zero. Moreover, the LOD value calculated for the G index was the lowest. A large slope of the variation curve and a low LOD indicated an increase in the method sensitivity.Consequently,color component G was selected as the color index because of the linearity, slope, and LOD.

    3.3. Stability of the PAD

    The initial screening experiments of several stabilizers were carried out. A higher component G value indicated a greater decrease in the activity of XOD. We tested the paper-based chips kept at room temperature for three days to screen for the appropriate stabilizer. It was observed that XOD with the tested stabilizers clearly lost activity. Then, the other two storage environments (4°C and -20°C) were used, and the results are shown in Fig. 4. These results showed that XOD with 10% glycerol maintained enzyme activity for 7 days, while the effects of the other stabilizers were unsatisfactory.

    Next, the stability of XOD with 10% glycerol for 21 days was tested, and the results are displayed in Fig. 5. When the paperbased chip was frozen at -20°C, the G values remained nearly unchanged.However,XOD lost its activity gradually when stored at room temperature or at 4°C without a stabilizer.Under 4°C storage conditions, XOD containing 10% glycerol could retain its activity without any obvious changes in color component values during 15 days.After 21 days,however,XOD activity was obviously decreased.Therefore,10% glycerol could help enhance the shelf life of paperbased chips preloaded with XOD to some extent.

    3.4. Parameter optimization and method validation

    The results of the definitive screening design experiments are shown in Table S1. The stepwise regression model of the color index was developed using the coded values of each parameter. For color component G, the equation was established asY=145.72-17.73X2-4.59X4-4.59X5-23.59X6-8.55X7-9.75X2X3with a determination coefficient(R2)of 0.9249 and an adjustedR2value of 0.8998. A highR2value means that most of the variations in the experimental data could be explained by the equation.Moreover,according to the coefficients of each factor in these two models,factors including 10%glycerol volume,NBTconcentration,PB solution volume,PB solutionpH and reaction time all showed negative correlations with the color index.

    The results of the validation experiments are listed in Table S2.The validation results proved that the experimental values of color component G were close to the predicted values. This finding indicated that the model had a good predictive ability. When less XOD and xanthine were added, the cost of the device would be decreased. Moreover, with a shorter reaction time and lower temperature, the efficiency of the method would be increased.Therefore,the optimal conditions were determined as follows:XOD concentration of 0.05 U/mL,10%glycerol volume of 15 μL,xanthine volume of 60 μL, NBT concentration of 1.5 mg/mL, PB solution volume of 25 μL, PB pH of 8.0, reaction time of 30 min, reaction temperature of 25°C, and drying temperature of 35°C.

    The XOD inhibitory activity of allopurinol solutions with different concentrations was measured with the optimized method. The range of calibration curve was 10-80 μg/mL. The G value and allopurinol concentration were found to be related as Y=1.2277X1-151.96.LOD and LOQ were calculated to be 10.9 μg/mL and 32.9 μg/mL,respectively.To further confirm the accuracy of the method, spike recovery experiments were performed at concentration levels of about 50%, 100%, and 150% of SME samples. The inhibitory effects of the SME samples were converted to allopurinol equivalents. As shown in Table S3, the average recoveries of samples were in the range between 87.06% and 109.95%, proving that the PAD method could achieve accurate results.

    4. Discussion

    4.1. Comparison with conventional methods

    Fig.5.Influence of 10% glycerol solution on the stability of XOD in three storage environments over 21 days. (A) Stored at room temperature, (B) stored at 4 °C, and (C)stored at -20 °C.

    PAD and the conventional microplate method was adopted to examine whether XOD was inhibited by SME samples.The SME was diluted with PB solution to 1/8-1/40 with three different concentrations. The color of samples was light. A blank control without XOD solution was added in each measurement. The color component values of samples were calculated by subtracting the values of blank control. In this way, PAD results would not be interfered by the color of tested samples. The results are shown in Fig. 6. There was no significant difference (Friedman test,P=0.368) among the three methods, which indicated the reliability of the PAD method.

    Fig.6.Comparison of the inhibitory effects of SME samples obtained with the microplate assay and PAD methods.

    4.2. Active component of SME and selectivity of the PAD method

    The results of the active component verification and selectivity experiments are shown in Fig.S5.The color component G values of PAD were measured,and the results of these four groups from left to right were 56,116, 99, and 59, respectively.

    The G values of SME group and salvianolic acid B solution group were both significantly higher than that of the blank control group.It shows that both the SME and salvianolic acid B solution can inhibit the XOD activity.In other words,salvianolic acid B is one of the active components of SME which can effectively inhibit XOD.It is consistent with the results reported in the literature [34]. There was no significant change in the color between mixed sugar solution group and the blank control group. It means that the mixed sugar solution has little effect on the XOD activity,which proves the selectivity of the PAD method proposed.

    4.3. Potential applications of the present technology

    There are many advantages of the conventional microplate method.For example,the repeatability of analysis results is usually good. Because the solutions used for microplate analysis is often prepared before use,there is no need to worry about the stability of the solutions at most occasions. However, a microplate reader is required to determine the results of microplate method. The microplate reader is usually not cheap, and inconvenient to move.These disadvantages limit the applications of microplate method for onsite analysis.

    In this work,the 3D-printed devices used in the PAD analysis were small.The size of the detection box was 9.0 cm×7.0 cm×11.5 cm,while the size of the reaction support device was 13.0 cm×3.5 cm×3.9 cm.Smartphones are very popular in China.Therefore, these PADs may be a powerful tool for the rapid quality testing of TCMs onsite during harvest and collection periods.Because the 3D-printed devices used in the PAD analysis are also cheap,the present method is promising in allowing users to conveniently assess the bioactivity of homemade herbal decoctions at home.The decision on harvesting a herbal material or taking a decoction can be made rapidly.Compared with conventional microplate method,microplate reader was not required, which further lowers the cost of analysis.Because enzyme, substrate, and chromogenic reagent are dried on PADs in advance,only sample solution and PB solution are required in theanalysis,which reduces theworkloadthrough adding lesskinds of solutions in the reaction system.

    SME is also the intermediate of many TCM preparations,such as Danshen injection. Quality analysis requiring long time is not suitable for industrial intermediates. The present method could also be used to control the quality of production intermediates of TCMs. Furthermore, PADs with certain enzymes immobilized on them for POC food bioactivity determination can be accessible.

    As a bioassay method,the advantage of the present PAD method is evaluating the TCM quality in general.However,the specificity of bioassay of TCMs is usually lower than that of chemical analysis methods, such as the chemical fingerprint method [6]. Therefore,bioassay is usually considered as a supplement to the current TCM quality evaluation mode [6]. When using the PAD method to determine TCM quality, the identification of medicinal herbs according to their appearance can improve the specificity.

    5. Conclusion

    An XOD activity assay method was developed using an optimized paper-based device.In this work,3D printing,paper cutting,and folding methods were used to create the double-layer structure PAD.The enzymatic reactions resulted in color intensity variations,which were recognized and distinguished by color component G.Next, a definitive screening design was employed to optimize the reaction conditions.The PAD method was proven to be accurate by the sample spike experiments. Under the optimal conditions, the effects of SME on XOD were tested by PAD with two different detection devices and the microplate method at the same time.The stability experiments showed that the PAD with immobilized enzymes could be stored for at least 15 days without a decrease in activity after adding 10% glycerol under 4°C or -20°C storage conditions.Salvianolic acid B in SME possessed the ability to inhibit XOD activity. Furthermore, all the reaction and detection devices were of small size,making them portable and easy for operators to carry and use. Thus, the PAD offers a POC platform for the convenient measurement of XOD inhibitory activity.The present method is promising in POC analysis.

    Declaration of competing interest

    The authors declare that there are no conflicts of interest.

    Acknowledgments

    The authors would like to thank the support of the National S&T Major Project of China (Grant No.: 2018ZX09201011), the National Natural Science Foundation of China (Grant No.: 81503242), and the Fundamental Research Funds for the Central Universities(Grant No.:2018FZA7018).

    Appendix A. Supplementary data

    Supplementary data to this article can be found online at https://doi.org/10.1016/j.jpha.2020.09.004.

    久久久国产成人精品二区| 欧美午夜高清在线| 日韩免费av在线播放| 99国产极品粉嫩在线观看| 神马国产精品三级电影在线观看| 日本 欧美在线| 男插女下体视频免费在线播放| 欧美三级亚洲精品| 桃红色精品国产亚洲av| 最新在线观看一区二区三区| 国产在视频线在精品| 91狼人影院| 免费看光身美女| 少妇熟女aⅴ在线视频| 久久精品91蜜桃| 国产三级在线视频| 国产真实伦视频高清在线观看 | 亚洲欧美清纯卡通| 亚洲狠狠婷婷综合久久图片| 国产高清有码在线观看视频| 一本久久中文字幕| 一级作爱视频免费观看| 欧美色欧美亚洲另类二区| 一级av片app| 热99re8久久精品国产| 久久人人精品亚洲av| 91字幕亚洲| 日韩免费av在线播放| 91午夜精品亚洲一区二区三区 | 给我免费播放毛片高清在线观看| 狠狠狠狠99中文字幕| 亚洲无线在线观看| 综合色av麻豆| 亚洲aⅴ乱码一区二区在线播放| 毛片一级片免费看久久久久 | 亚洲黑人精品在线| 亚洲自偷自拍三级| 国产精品一区二区三区四区久久| 免费av毛片视频| 国产精品一及| 国产一区二区激情短视频| 深爱激情五月婷婷| 高清毛片免费观看视频网站| aaaaa片日本免费| 老司机福利观看| 日日摸夜夜添夜夜添小说| 免费无遮挡裸体视频| 久久精品人妻少妇| 国产精品影院久久| 3wmmmm亚洲av在线观看| 国产精品免费一区二区三区在线| 少妇丰满av| 国产一区二区亚洲精品在线观看| 在线观看一区二区三区| 五月玫瑰六月丁香| 午夜福利免费观看在线| 91在线精品国自产拍蜜月| 久99久视频精品免费| 桃红色精品国产亚洲av| 欧美乱妇无乱码| 欧美乱妇无乱码| 久久久色成人| 极品教师在线视频| 国产又黄又爽又无遮挡在线| eeuss影院久久| 亚洲专区中文字幕在线| 99视频精品全部免费 在线| 夜夜看夜夜爽夜夜摸| 日韩免费av在线播放| 亚洲av成人精品一区久久| 国产伦在线观看视频一区| 国产在线精品亚洲第一网站| 国产又黄又爽又无遮挡在线| 日日摸夜夜添夜夜添av毛片 | 久久精品国产自在天天线| 老熟妇仑乱视频hdxx| 亚洲av免费高清在线观看| 在现免费观看毛片| 国产亚洲欧美98| 国产在线精品亚洲第一网站| 国产精品99久久久久久久久| 婷婷精品国产亚洲av| 亚洲 欧美 日韩 在线 免费| 亚洲美女黄片视频| 亚洲综合色惰| 精品久久久久久久久av| 麻豆成人av在线观看| 观看美女的网站| 淫秽高清视频在线观看| 成人午夜高清在线视频| 久久久久国内视频| 国产欧美日韩一区二区精品| 久久草成人影院| 久久精品国产亚洲av天美| 又黄又爽又刺激的免费视频.| 很黄的视频免费| 人妻久久中文字幕网| 最新在线观看一区二区三区| 亚洲精品乱码久久久v下载方式| 成人av在线播放网站| 99热6这里只有精品| 99热这里只有精品一区| 欧美国产日韩亚洲一区| 天堂影院成人在线观看| 国产精品电影一区二区三区| 免费人成在线观看视频色| 国产精品野战在线观看| 欧美精品啪啪一区二区三区| 又黄又爽又免费观看的视频| 在线a可以看的网站| 国产老妇女一区| 99国产精品一区二区三区| 小说图片视频综合网站| 日韩免费av在线播放| 草草在线视频免费看| 18禁黄网站禁片免费观看直播| 亚洲 国产 在线| 日韩免费av在线播放| 两个人的视频大全免费| 久久精品人妻少妇| 日韩成人在线观看一区二区三区| 美女大奶头视频| 日本五十路高清| 亚洲一区二区三区色噜噜| 亚洲精品一卡2卡三卡4卡5卡| 在线观看免费视频日本深夜| 亚洲中文字幕日韩| 中文字幕av成人在线电影| 老熟妇仑乱视频hdxx| 久久精品久久久久久噜噜老黄 | 色播亚洲综合网| 久久精品综合一区二区三区| 少妇丰满av| 嫩草影视91久久| 亚洲 欧美 日韩 在线 免费| 一级黄色大片毛片| 国产精品国产高清国产av| 人妻夜夜爽99麻豆av| 精华霜和精华液先用哪个| 久久国产乱子伦精品免费另类| 国产精品影院久久| 日韩欧美三级三区| 亚洲av成人av| 91久久精品国产一区二区成人| 中国美女看黄片| 免费在线观看亚洲国产| 最近最新中文字幕大全电影3| 色综合婷婷激情| 在线观看免费视频日本深夜| 五月伊人婷婷丁香| 我要看日韩黄色一级片| a级毛片免费高清观看在线播放| 日韩欧美免费精品| 亚洲五月天丁香| 一级黄片播放器| www.熟女人妻精品国产| 99精品在免费线老司机午夜| 亚洲成人免费电影在线观看| 午夜福利在线观看免费完整高清在 | 亚洲电影在线观看av| 国产一区二区三区视频了| 日韩精品青青久久久久久| 男女下面进入的视频免费午夜| 国产91精品成人一区二区三区| 香蕉av资源在线| 日日干狠狠操夜夜爽| 国产真实乱freesex| 欧美极品一区二区三区四区| 欧美潮喷喷水| 亚洲va日本ⅴa欧美va伊人久久| 看黄色毛片网站| 在线a可以看的网站| 婷婷精品国产亚洲av在线| 久久久久久久久大av| 欧美日韩中文字幕国产精品一区二区三区| 日韩欧美免费精品| 日本一二三区视频观看| 国产精品伦人一区二区| 首页视频小说图片口味搜索| 精品久久久久久久久久久久久| 啦啦啦观看免费观看视频高清| 在线国产一区二区在线| 在线观看66精品国产| av在线蜜桃| 精品人妻视频免费看| 露出奶头的视频| 国产美女午夜福利| 人妻丰满熟妇av一区二区三区| 99热精品在线国产| 精品一区二区三区视频在线观看免费| 色哟哟哟哟哟哟| 男女下面进入的视频免费午夜| 丰满乱子伦码专区| 国模一区二区三区四区视频| 国产69精品久久久久777片| 乱码一卡2卡4卡精品| 人妻制服诱惑在线中文字幕| 欧美+亚洲+日韩+国产| 免费av毛片视频| 成人午夜高清在线视频| 校园春色视频在线观看| 在线播放无遮挡| 一进一出抽搐gif免费好疼| 九九久久精品国产亚洲av麻豆| 他把我摸到了高潮在线观看| 色精品久久人妻99蜜桃| 简卡轻食公司| av专区在线播放| 国产老妇女一区| 精品午夜福利在线看| 97热精品久久久久久| 蜜桃亚洲精品一区二区三区| 看片在线看免费视频| 午夜福利在线观看免费完整高清在 | 久久久久免费精品人妻一区二区| 免费看光身美女| 国产精品久久久久久亚洲av鲁大| 精品久久国产蜜桃| 性色avwww在线观看| 美女高潮的动态| 日韩欧美国产在线观看| 国产精品国产高清国产av| 亚洲精品亚洲一区二区| 综合色av麻豆| 最近最新免费中文字幕在线| 免费看日本二区| 脱女人内裤的视频| 波野结衣二区三区在线| av欧美777| 成人午夜高清在线视频| 成熟少妇高潮喷水视频| 欧美+日韩+精品| 亚洲成人精品中文字幕电影| 99国产综合亚洲精品| 热99re8久久精品国产| netflix在线观看网站| 夜夜看夜夜爽夜夜摸| 久久久久久久久大av| 免费人成视频x8x8入口观看| 免费看日本二区| 久久久久久久久中文| 午夜福利在线在线| 免费看美女性在线毛片视频| 日本撒尿小便嘘嘘汇集6| 亚洲中文日韩欧美视频| 久久久久亚洲av毛片大全| 长腿黑丝高跟| 真人一进一出gif抽搐免费| 国产成人a区在线观看| 亚洲 欧美 日韩 在线 免费| 88av欧美| 好男人在线观看高清免费视频| 99久久成人亚洲精品观看| 欧美最新免费一区二区三区 | 亚洲无线在线观看| 亚洲人与动物交配视频| 精品久久久久久久久久免费视频| 内射极品少妇av片p| 国产三级在线视频| 午夜亚洲福利在线播放| 色综合欧美亚洲国产小说| 黄色配什么色好看| 成人国产一区最新在线观看| 亚洲最大成人av| 国产亚洲精品久久久com| 久久久久精品国产欧美久久久| 亚洲经典国产精华液单 | 成人鲁丝片一二三区免费| 9191精品国产免费久久| 久久午夜亚洲精品久久| 亚洲人与动物交配视频| 国产av麻豆久久久久久久| 好男人电影高清在线观看| 综合色av麻豆| 久久99热6这里只有精品| 91在线观看av| 国产精品影院久久| 69人妻影院| 亚洲欧美精品综合久久99| 熟妇人妻久久中文字幕3abv| 久久国产乱子伦精品免费另类| 久久久久久国产a免费观看| 国产成人av教育| 免费av观看视频| 免费无遮挡裸体视频| 久久中文看片网| 狂野欧美白嫩少妇大欣赏| 日韩国内少妇激情av| 亚洲片人在线观看| 毛片女人毛片| 看黄色毛片网站| 国产av在哪里看| 最近视频中文字幕2019在线8| 日本一二三区视频观看| 天堂av国产一区二区熟女人妻| 国产精品久久久久久久电影| 波多野结衣高清作品| 在现免费观看毛片| 午夜日韩欧美国产| 色噜噜av男人的天堂激情| 欧美成狂野欧美在线观看| 午夜视频国产福利| 亚洲人成网站在线播放欧美日韩| 欧美午夜高清在线| 99riav亚洲国产免费| 蜜桃久久精品国产亚洲av| 在线a可以看的网站| 哪里可以看免费的av片| 非洲黑人性xxxx精品又粗又长| 国产在线男女| 少妇人妻一区二区三区视频| 久久久久九九精品影院| 精品久久久久久久人妻蜜臀av| ponron亚洲| av视频在线观看入口| 高潮久久久久久久久久久不卡| 国产乱人视频| 欧美激情在线99| 亚洲三级黄色毛片| 欧美又色又爽又黄视频| 69人妻影院| 夜夜躁狠狠躁天天躁| 夜夜看夜夜爽夜夜摸| 亚洲av电影在线进入| 亚洲av电影不卡..在线观看| 又黄又爽又免费观看的视频| 精品乱码久久久久久99久播| 日韩亚洲欧美综合| 熟妇人妻久久中文字幕3abv| 91麻豆精品激情在线观看国产| 精品欧美国产一区二区三| 午夜日韩欧美国产| 国产精品av视频在线免费观看| 青草久久国产| 99久久精品一区二区三区| 美女免费视频网站| a在线观看视频网站| 国产色婷婷99| av中文乱码字幕在线| 嫩草影院入口| 国产精品人妻久久久久久| 五月伊人婷婷丁香| 久久久色成人| 91狼人影院| 国产精品久久久久久精品电影| 中亚洲国语对白在线视频| 淫妇啪啪啪对白视频| 午夜福利高清视频| 国产精品爽爽va在线观看网站| 国产成人欧美在线观看| 一级黄色大片毛片| 九九热线精品视视频播放| 国产 一区 欧美 日韩| 91字幕亚洲| 亚洲精品影视一区二区三区av| 啦啦啦韩国在线观看视频| 国产精品久久久久久亚洲av鲁大| 黄色女人牲交| 男女做爰动态图高潮gif福利片| 91在线精品国自产拍蜜月| 亚洲欧美清纯卡通| 在线国产一区二区在线| .国产精品久久| 直男gayav资源| 国内久久婷婷六月综合欲色啪| 女人被狂操c到高潮| 天堂网av新在线| 麻豆一二三区av精品| 不卡一级毛片| 亚洲国产精品999在线| 一进一出好大好爽视频| 日韩高清综合在线| 欧美+日韩+精品| 国产精品爽爽va在线观看网站| 国产淫片久久久久久久久 | 一卡2卡三卡四卡精品乱码亚洲| 日本黄色视频三级网站网址| 最近中文字幕高清免费大全6 | 一个人观看的视频www高清免费观看| 亚洲在线观看片| 亚洲美女视频黄频| 一级作爱视频免费观看| 黄色日韩在线| 五月玫瑰六月丁香| 国产蜜桃级精品一区二区三区| 真实男女啪啪啪动态图| 欧美在线黄色| 桃红色精品国产亚洲av| 成人高潮视频无遮挡免费网站| 亚洲精品成人久久久久久| 国产精品嫩草影院av在线观看 | 久久久久久久亚洲中文字幕 | 午夜精品久久久久久毛片777| 欧美日韩瑟瑟在线播放| 国产国拍精品亚洲av在线观看| 久久久久性生活片| 精品国产亚洲在线| 日日干狠狠操夜夜爽| 欧美潮喷喷水| 日韩欧美三级三区| 观看美女的网站| 成人一区二区视频在线观看| 色在线成人网| 久久天躁狠狠躁夜夜2o2o| 欧美日本视频| 国产精品免费一区二区三区在线| 青草久久国产| 欧美不卡视频在线免费观看| 亚洲狠狠婷婷综合久久图片| 51国产日韩欧美| 亚洲成人免费电影在线观看| 我要看日韩黄色一级片| 夜夜躁狠狠躁天天躁| 91av网一区二区| 99热6这里只有精品| 999久久久精品免费观看国产| 天堂√8在线中文| 内地一区二区视频在线| 嫩草影视91久久| 91久久精品国产一区二区成人| 亚洲三级黄色毛片| 免费av毛片视频| 免费看日本二区| 悠悠久久av| 69av精品久久久久久| 免费av观看视频| 精品久久国产蜜桃| 十八禁国产超污无遮挡网站| 亚洲乱码一区二区免费版| 久9热在线精品视频| 老熟妇乱子伦视频在线观看| 日本精品一区二区三区蜜桃| 如何舔出高潮| 久久国产乱子伦精品免费另类| 国产亚洲精品久久久com| 成人高潮视频无遮挡免费网站| 美女高潮喷水抽搐中文字幕| 中文字幕免费在线视频6| 丁香欧美五月| 免费在线观看亚洲国产| 免费在线观看影片大全网站| 一边摸一边抽搐一进一小说| 小蜜桃在线观看免费完整版高清| 久久精品国产清高在天天线| 成人性生交大片免费视频hd| 999久久久精品免费观看国产| 嫩草影院入口| 免费无遮挡裸体视频| 性色av乱码一区二区三区2| 少妇的逼水好多| 变态另类丝袜制服| 久9热在线精品视频| 动漫黄色视频在线观看| 中国美女看黄片| 精品福利观看| 欧美激情在线99| 久久精品国产亚洲av涩爱 | 亚洲五月天丁香| 亚洲国产精品合色在线| 国产精品三级大全| 国产色婷婷99| 日韩欧美三级三区| 黄色丝袜av网址大全| 在线观看一区二区三区| 首页视频小说图片口味搜索| 久久久精品大字幕| 啪啪无遮挡十八禁网站| 人人妻人人看人人澡| 久久精品国产清高在天天线| 亚洲男人的天堂狠狠| 无遮挡黄片免费观看| 别揉我奶头~嗯~啊~动态视频| 国产成人av教育| 两个人的视频大全免费| 在线观看美女被高潮喷水网站 | 人妻制服诱惑在线中文字幕| 国产成人福利小说| 成人美女网站在线观看视频| 国产精品免费一区二区三区在线| 两人在一起打扑克的视频| 亚洲一区二区三区色噜噜| 一本久久中文字幕| 国产精品1区2区在线观看.| 听说在线观看完整版免费高清| 国产在线精品亚洲第一网站| 亚洲片人在线观看| 老司机福利观看| www日本黄色视频网| 久久精品国产99精品国产亚洲性色| 精品一区二区三区人妻视频| 亚洲精品影视一区二区三区av| 国产熟女xx| 一进一出好大好爽视频| 午夜福利成人在线免费观看| 国产野战对白在线观看| 亚洲人成电影免费在线| 夜夜看夜夜爽夜夜摸| 日韩高清综合在线| av天堂中文字幕网| 69av精品久久久久久| 两性午夜刺激爽爽歪歪视频在线观看| 精品久久国产蜜桃| 欧美一级a爱片免费观看看| 精品乱码久久久久久99久播| www.熟女人妻精品国产| 3wmmmm亚洲av在线观看| 男人狂女人下面高潮的视频| 日本与韩国留学比较| bbb黄色大片| 国产乱人视频| 丰满乱子伦码专区| 日韩大尺度精品在线看网址| 欧美黑人欧美精品刺激| 最近最新中文字幕大全电影3| 精品久久久久久久久亚洲 | 欧美丝袜亚洲另类 | 91字幕亚洲| 99精品久久久久人妻精品| 精品一区二区三区人妻视频| 久久久色成人| 国产免费一级a男人的天堂| 真人一进一出gif抽搐免费| 国产一区二区三区视频了| 国模一区二区三区四区视频| av福利片在线观看| 亚洲五月天丁香| 国产黄片美女视频| 色播亚洲综合网| 国语自产精品视频在线第100页| 噜噜噜噜噜久久久久久91| 91在线观看av| 国产精品自产拍在线观看55亚洲| 夜夜躁狠狠躁天天躁| 少妇的逼好多水| 波多野结衣巨乳人妻| or卡值多少钱| 国产熟女xx| 免费观看人在逋| 国产精品99久久久久久久久| 午夜亚洲福利在线播放| 一个人看视频在线观看www免费| 九九热线精品视视频播放| 亚洲中文日韩欧美视频| 欧美性猛交黑人性爽| 天堂av国产一区二区熟女人妻| 亚洲18禁久久av| 国产精品电影一区二区三区| 欧美一区二区国产精品久久精品| 国产色婷婷99| 色哟哟·www| 丁香六月欧美| 亚洲熟妇熟女久久| 国内精品久久久久精免费| 天堂√8在线中文| 日韩欧美在线乱码| 欧美一区二区亚洲| 日韩精品中文字幕看吧| 日本 欧美在线| 亚洲 欧美 日韩 在线 免费| 亚洲欧美日韩高清专用| 一进一出抽搐gif免费好疼| 亚洲人与动物交配视频| 日韩欧美精品v在线| 夜夜夜夜夜久久久久| av在线观看视频网站免费| 99热6这里只有精品| 搡老岳熟女国产| 久久伊人香网站| 欧美黑人欧美精品刺激| 国产欧美日韩精品一区二区| 九九热线精品视视频播放| 国产精品亚洲美女久久久| 亚洲av电影在线进入| 国产成+人综合+亚洲专区| 91在线精品国自产拍蜜月| 又黄又爽又免费观看的视频| 久久午夜福利片| 国产老妇女一区| 在现免费观看毛片| 国产精品久久久久久亚洲av鲁大| 欧美成人一区二区免费高清观看| 国语自产精品视频在线第100页| 老鸭窝网址在线观看| 一级黄色大片毛片| 国产亚洲精品久久久com| 日韩免费av在线播放| 老司机午夜十八禁免费视频| 少妇被粗大猛烈的视频| 老师上课跳d突然被开到最大视频 久久午夜综合久久蜜桃 | or卡值多少钱| 欧美激情久久久久久爽电影| 久久久久国内视频| 999久久久精品免费观看国产| 国产淫片久久久久久久久 | 国产精品免费一区二区三区在线| 老女人水多毛片| 琪琪午夜伦伦电影理论片6080| 午夜视频国产福利| 国产高清有码在线观看视频| 黄色一级大片看看| 亚洲欧美清纯卡通| 午夜福利高清视频| 成年免费大片在线观看| 亚洲成人精品中文字幕电影| 国产精品永久免费网站| 国产一区二区在线观看日韩| 中文字幕人妻熟人妻熟丝袜美| 一进一出好大好爽视频| 男女那种视频在线观看| 久久久久久久午夜电影| 日韩国内少妇激情av| 亚洲五月天丁香| 成人av一区二区三区在线看| 亚洲人成电影免费在线| 成人三级黄色视频|